Life expectancy in the industrialized world has increased dramatically over the past century. A consequence of the increasing life expectancy is that diseases generally associated with aging, including cancer and neurodegeneration, will affect more individuals. The genetic basis of cancer is attributed to a series of DNA damage events that can include point mutations, chromosome rearrangements and loss, and epigenetic changes such as altered DNA cytosine methylation patterns. DNA is known to be damaged by exogenous chemicals and radiation. However, cellular DNA is continuously oxidized, hydrolyzed and methylated even in the absence of exogenous agents. It is estimated that the number of endogenous DNA lesions formed is on the order of tens of thousands per cell per day. In response to DNA damage, cells have a complex network of repair pathways that recognize, excise and rebuild the damaged sites. Defects in DNA repair pathways are known to increase substantially the incidence of cancer. An increase in human life span over which DNA can be damaged, coupled with an increase in age-associated diseases including cancer, highlight the need to understand how DNA can be damaged and how repair pathways function.The focus of our efforts on this project has been five pyrimidine oxidation products that can result from radiation, carcinogen exposure and endogenous DNA damage. These modified pyrimidines include 5-hydroxymethyluracil (HmU), 5-formyluracil (FoU), 5-hydroxymethylcytosine (HmC), 5-hydroxyuracil (HoU) and 5-hydroxycytosine (HoC). In the previous period of funding, we discovered several important chemical properties of these modified pyrimidines, and we have discovered several new and significant properties of damaged base recognition by cellular repair enzymes.In the current proposal, we describe a series of experiments to follow up on these findings. The specific aims of the current proposal are 1) to identify the proteins responsible for the repair of HmU and to further understand cellular responses to the repair of exogenous HmU, 2) to investigate biochemical consequences of the oxidation of 5- methylcytosine (5mC) including the impact of HmC on DNA-protein interactions and to further investigate a potential novel biochemical pathway through which methylation patterns might be altered, 3) to identify the general mechanisms by which damaged pyrimidines are recognized by DNA repair glycosylases, and 4) to develop a general strategy for the identification and characterization of as yet unidentified DNA repair proteins. The results of the proposed studies should shed significant new light on our understanding of DNA damage and its repair. With these added insights, we may learn how to reduce DNA damage that leads to human diseases including cancer, as well as understand how to exploit defects or alterations in DNA repair pathways for the development of more selective chemotherapy.